Molecular Epidemiology of Plasmodium falciparum Chloroquine Resistance Transporter Genes among School Children in Kwara State, Southwestern Nigeria

Background: Plasmodium falciparum existence continues to develop resistance to conventional antimalaria drugs in malaria endemic areas. Plasmodiaoften prevent drugs from interacting with the target site, hence, developing resistance to antimalaria drugs. Mutations in the Plasmodium falciparum chloroquine resistance transporter (Pfcrt), are the major determinant of chloroquine resistance in human malaria parasite.Methodology:Malaria infection, Pfcrtand Pfmdr1 genes of isolates among school students within the age range of 11-22 years from four selected rural communities of Kwara state were studied.One hundred and eighty seven subjects (187) wereselected for the study. Blood samples were collected by finger prick method for malaria screening. Nested PCR and restriction fragment length polymorphism (RFLP) were done to detect alleles of pfcrtat codon 76 and pfmdr1at codon 86. DNA of isolates wasappropriately extracted from the filter paper blots using the methanol fixation method. Logistic regression was performed on the binary observations obtained while linear regression was conducted on the fifty (50) subjects that tested positive to malaria.Results:Out of 187 subjects screened, 26.7% (50) were positive to P. falciparum. Highest malaria parasite count of 36.4% was recorded in 14-16years age group while 20-22 years age group had the least malaria parasite count (15.4%). The result of the studied isolates indicated that out of 50 isolates analyzed for Pfcrtgene, wild type alleles accounted for 32% (16) while mutant alleles accounted for 68% (34). Alakuko Community accounted for the least number of T76 mutant alleles 10% (5) while Apado community recorded the highest number of T76 mutant gene 22% (11). For Pfmdr1gene analysis at codon 86, isolates from Apado community showed the highest mutant type alleles (Y86) of 22% (11), while Igbonla community in Ifelodun local government had the least mutant alleles, 6% (3).Conclusion:The overall result revealed existence of mutant alleles in both the Pfcrtand Pfmdr1genes which was higher than the wild type gene in both cases. The presence of chloroquine resistance genes among the studied population implies that alternative antimalaria drugs should be designed by pharmaceutical industry.

Genetic Polymorphisms of Pfcrt K76T and Pfmdr1 N86Y among Asymptomatic School Children in Forest Communities of Ekondo Titi Subdivision along the Cameroon-Nigeria Border Area

Aims:The study sought to quantifyPlasmodiuminfection and molecular markers for chloroquine resistance among asymptomatic school children.Study Design:The study was cross-sectional.Place and Duration of Study:The study was carried out in Ekondo Titi Subdivision near Original Research Article Cameroon's south-western border with Nigeria from March to May and from September to October 2014.Methodology:The prevalence of humanPlasmodium specieswas determined by nested PCR (Polymerase Chain Reaction) using DNA from dried blood spot in six primary schools. A PCR/RFLP analysis (Restriction Fragment Length Polymorphism) was used to determine the prevalence of chloroquine resistance (CQR) associatedpfcrt76T andpfmdr1 56Y point mutations in Plasmodiumfalciparumasymptomatic school children.Results: A nested PCR amplifying the 18S small-subunit ribosomal RNA (SSU rRNA) gene of Plasmodiumin 205 samples confirmed 76.1% of the isolates as asymptomatic P.falciparuminfections, with a substantial proportion 22% ofP. malariaeinfection. Among these, 3.6% were singleP. malariaeinfections and 15.1% wereP. falciparumandP. malariaemixed infections. MixedP. falciparumandP. ovaleinfections were2.0%. Of the 156Plasmodium falciparum, positive samples by species-specific PCR, 107 samples withP. falciparummono-infection were analyzed for the presence of drug resistant allelespfcrt76T andpfmdr1-Y 86. The prevalence ofpfcrt76T mutation (74.6%) was higher than that of thepfmdr1-Y86 mutation (25.4%). Logistic regression analysis of socio-demographic factors predicted no significant association betweenpfcrt76T mutation with gender and communities.Conclusions:The results indicated a high prevalence ofP. malariaeand mixed infection in the area under study. The high-level distribution of thepfcrtT76observed in the study could be possibly attributed to the fact that CQ remained widely used at the community level more than 14 years after withdrawal

Pfcrt K76T and Pfmdr 1 Resistance Genes in Post Chloroquine Era in South-South Region of Nigeria

Malaria, a global health problem especially in the sub-Sahara region has been posing a recurrent problem due to the resistance of the parasites to the available antimalarial drugs despite the preventive measures provided by WHO.Aims:This study is aimed at determining the prevalence of resistance markers in four Niger Delta states of Nigeria, a decade after withdrawal of chloroquine.Methods:Eight hundred and forty six (846) subjects participated in the study and were distributed as follows, 192(22.7%) Bayelsa; 218(25.8%) Rivers; 196(23.2%) Edo and 240(28.4%) Delta respectively. Malaria parasite identification was carried out using standard parasitological techniques. Genotyping of the resistance markers Pfcrt K76T and Pfmdr 1 was carried out by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP).Results:Our findings revealed that the prevalence of malaria infection in the four Niger Delta states were 78.1%, 68.8%, 62.8% and 58.8% in Bayelsa, Rivers States, Edo and Delta respectively. There was no statistical difference in the prevalence of malaria within the four Niger Delta states. (P>0.05). Children below the age of 5 years recorded the highest infection rates when compared to subjects in other age groups (P< 0.01). Our findings also revealed that the distribution of mutant Pfcrt K76T and Pfmdr 1 genes across the four states were 12.0% and 28.6%, 4.0% and 22.0%, 14.6% and 29.3%, 10.6% and 25.0% in Bayelsa, Rivers, Edo and Delta state respectively. However, the prevalence of Pfcrt K76T in Rivers State was statistically lower when compared to other states (P < 0.01) while no statistical difference existed in the distribution of Pfmdr 1 mutant genes (P>0.01).Conclusion:Prevalenceof Pfcrt and Pfmdr 1 remained elevated in the Niger Delta states despite the withdrawal of chloroquine over a decade ago. Hence, Nigeria is far from an eventual re-introduction of chloroquine as its resistance markers still persist in our communities. Furthermore, the root cause of the persistence of these resistance markers needs to be investigated

Molecular Surveillance of Pfkelch13 and Pfmdr1 Mutations in Plasmodium falciparum Isolates from Southern Thailand

Artemisinin-based combination therapy (ACT) resistance is widespread throughout the Greater Mekong Subregion. This raises concern over the antimalarial treatment in Thailand since it shares borders with Cambodia, Laos, and Myanmar where high ACT failure rates were reported. It is crucial to have information about the spread of ACT resistance for efficient planning and treatment. This study was to identify the molecular markers for antimalarial drug resistance: Pfkelch13 and Pfmdr1 mutations from 5 provinces of southern Thailand, from 2012 to 2017, of which 2 provinces on the Thai- Myanmar border (Chumphon and Ranong), one on Thai-Malaysia border (Yala) and 2 from non-border provinces (Phang Nga and Surat Thani). The results showed that C580Y mutation of Pfkelch13 was found mainly in the province on the Thai-Myanmar border. No mutations in the PfKelch13 gene were found in Surat Thani and Yala. The Pfmdr1 gene isolated from the Thai-Malaysia border was a different pattern from those found in other areas (100% N86Y) whereas wild type strain was present in Phang Nga. Our study indicated that the molecular markers of artemisinin resistance were spread in the provinces bordering along the Thai-Myanmar, and the pattern of Pfmdr1 mutations from the areas along the international border of Thailand differed from those of the non-border provinces. The information of the molecular markers from this study highlighted the recent spread of artemisinin resistant parasites from the endemic area, and the data will be useful for optimizing antimalarial treatment based on regional differences.

Prevalence of Pfmdr 1 N86Y and Y184F Alleles is Associated with Recurrent Parasitemia following Treatment of Uncomplicated Malaria with Artemether-Lumefantrine in Nigerian Patients.

We investigated and compared genetic variations in Plasmodium falciparum multidrug resistance 1 gene (Pfmdr 1) in patients showing good therapeutic response (GTR) and artemisinin resistance (AR) following artemether-lumefantrine (AL) treatment of uncomplicated malaria in Nigeria. Some 150 malaria patients were subjected to AL treatment and therapeutic efficacy was monitored for 28 days. Parasite genomic DNA was isolated followed by nested polymerase chain reaction (PCR). Genotyping of Pfmdr 1 gene for specific genetic variants: N86Y, Y184F, S1034C and N1042D were done using PCR-restriction fragment length polymorphism (PCR-RFLP).Out of 121 patients that were P. falciparum positive, 46 % (56) and 54 % (65) showed good therapeutic response and artemisinin resistance respectively, with 5.4 % and 98.3 % being mutated in the GTR and AR group respectively. The most prevalent mutations were Y184F (44.1 %) and N86Y (40.7 %). There was significant increase (p<0.001) in the prevalence of Pfmdr 1 mutation in the post treatment compared to the pre-treatment group.Prevalence of Pfmdr1 86Y and 184F alleles is associated with artemisinin resistance and presence of AL drug significantly induced genetic variation in the plasmodial gene.

Identification of chloroquine resistance Pfcrt-K76T and determination of Pfmdr1-N86Y copy number by SYBR Green I qPCR

Objective: To identify prevalence of chloroquine resistance point mutation at (Pfcrt, K76T) and (Pfmdr1, N86Y) copy number variation. Methods: SYBR Green I based real time PCR was used. One hundred and thirty-three samples were analyzed for (Pfcrt, K76T) and (Pfmdr1,N86Y) copy number from dried blood spot. Parasite DNA was extracted using high pure DNA preparation kit. The amplification of DNA was done by using AccuPower 2× GreenStar

Polymorphic patterns of pfcrt and pfmdr1 in Plasmodium falciparum isolates along the Thai-Myanmar border

Objective: To investigate the distribution and patterns of pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum (P. falciparum) isolates collected from the malaria endemic area of Thailand along Thai-Myanmar border. Methods: Dried blood spot samples were collected from 172 falciparum malaria patients prior received treatment. The samples were extracted using chelex to obtain parasite DNA. PCR-RFLP was employed to detect pfcrt mutation at codons 76, 220, 271, 326, 356 and 371, and the pfmdr1 mutation at codon 86. Pfmdr1 gene copy number was determined by SYBR Green I real-time PCR. Results:Mutant alleles of pfcrt and wild type allele of pfmdr1 were found in almost all samples. Pfmdr1 gene copy number in isolates collected from all areas ranged from 1.0 to 5.0 copies and proportion of isolates carrying>1 gene copies was 38.1%. The distribution and patterns of pfcrt and pfmdr1 mutations were similar in P. falciparum isolates from all areas. However, significant differences in both number of pfmdr1 copies and prevalence of isolates carrying>1 gene copies were observed among isolates collected from different areas. The median pfmdr1 copy number in P. falciparum collected from Kanchanaburi and Mae Hongson were 2.5 and 2.0, respectively and more than half of the isolates carried>1 gene copies.

Sequence analysis of coding DNA fragments of pfcrt and pfmdr-1 genes in Plasmodium falciparum isolates from Odisha, India

The global emergence and spread of malaria parasites resistant to antimalarial drugs is the major problem in malaria control. The genetic basis of the parasite's resistance to the antimalarial drug chloroquine (CQ) is well-documented, allowing for the analysis of field isolates of malaria parasites to address evolutionary questions concerning the origin and spread of CQ-resistance. Here, we present DNA sequence analyses of both the second exon of the Plasmodium falciparum CQ-resistance transporter (pfcrt) gene and the 5' end of the P. falciparum multidrug-resistance 1 (pfmdr-1) gene in 40 P. falciparum field isolates collected from eight different localities of Odisha, India. First, we genotyped the samples for the pfcrt K76T and pfmdr-1 N86Y mutations in these two genes, which are the mutations primarily implicated in CQ-resistance. We further analyzed amino acid changes in codons 72-76 of the pfcrt haplotypes. Interestingly, both the K76T and N86Y mutations were found to co-exist in 32 out of the total 40 isolates, which were of either the CVIET or SVMNT haplotype, while the remaining eight isolates were of the CVMNK haplotype. In total, eight nonsynonymous single nucleotide polymorphisms (SNPs) were observed, six in the pfcrt gene and two in the pfmdr-1 gene. One poorly studied SNP in the pfcrt gene (A97T) was found at a high frequency in many P. falciparum samples. Using population genetics to analyze these two gene fragments, we revealed comparatively higher nucleotide diversity in the pfcrt gene than in the pfmdr-1 gene. Furthermore, linkage disequilibrium was found to be tight between closely spaced SNPs of the pfcrt gene. Finally, both the pfcrt and the pfmdr-1 genes were found to evolve under the standard neutral model of molecular evolution.

In vitro Potentiation of Chloroquine Activity in Plasmodium falciparum by Ketotifen and Cyproheptadine / 中国寄生虫学与寄生虫病杂志

Objective To study the potentiation of chloroquine activity and mechanism by ketotifen and cyproheptadine in in vitro cultured Plasmodium falciparum Fcc SM1/yN strain. Methods In vitro cultured Fcc SM1/yN strain was added to pre-prepared drug plates at 50 ?l/well after synchronization to make final concentration of 0.312 5-2 560 nmol/L for chloroqine and of 9.80-5 000 nmol/L for ketotifen or cyproheptadine. After 34 hours' culture in 37 ℃, the number of schizonts with 3 or more nuclei was calculated among 200 parasites under microscope. Calculated half inhibitive concentration ( IC50 ) of chloroquine and every drug combination to parasite as well as chloroquine activity enhancement index ( AEI) of ketotifen (or cyproheptadin) . Time dependency of potentiation was studied. All data were analyzed statistically with SPSS 13.0. After 20 hours' action of one optimal combination dose of chloroquine/ketotifen or chloroquine/cyproheptadine, RNA of the Fcc SM1/yN strain was extracted and real-time PCR was used to determine the expression level of pfcrt and pfmdr1 genes. Results The best potentiation effect was observed with ketotifen or cyproheptadine of 625 nmol/L, with IC50 of 74.53 nmol/L for chloroquine/ketotifen and 89.7 nmol/L for chloroquine/cyproheptadine respectively, and activity enhancement index (AEI) of 0.42 for chloroquine/ketotifen and 0.30 for chloroquine/cyproheptadine respectively. Combination of 625 nmol/Lketotifen or cyproheptadine with 5 nmol/L chloroquine showed the highest potentiation potency. 6-7 hours during which ketotifen or cyproheptadine was added after chloroquine showed the highest effect, with IC50 of 67.70 nmol/L for chloroquine/ketotifen and 81.53 nmol/L for chloroquine/cyproheptadine respectively, and the AEI was 0.47 for chloroquine/ketotifen and 0.37 for chloroquine/cyproheptadine respectively. After action of chloroquine/ketotifen or chloroquine/ cyproheptadine at one optimal combination dose, expression level of pfcrt gene increased by 91% and that of pfmdr1 gene decreased by 14% respectively. Conclusion Appropriate combination of chloroquine/ketotiphen or chloroquine/ cyproheptadine potentiates chloroquine against in vitro cultured P. falciparum. 6-7 hour period is an optimal time when ketotifen or cyproheptadine was added after chloroquine. Potentiating activity of ketotifen and cyproheptadine may be related to the expression level of pfcr t and pfmdr1 genes.